Globin genes in the beta-globin gene cluster are expressed, in humans and other vertebrates, in a developmental pattern. Abnormalities of human adult beta-globin gene expression, as in sickle cell disease or beta-thalassemia, have caused significant morbidity and mortality; a concurrent genetic persistence of fetal globin gene expression, however, is clinically ameliorative and has prompted a search for pharmacologic methods of up-regulating fetal/embryonic globin gene expression. This proposal describes an investigation into the molecular mechanisms of embryonic globin gene regulation, both developmentally and following pharmacologic manipulation. Earlier work, by this investigator, the sponsor, and other laboratories has confirmed the ability of short-chain fatty acids (SCFAs) to up-regulate embryonic and fetal globin gene expression in vivo and in vitro, and has implicated cis-acting sites. Here, we propose to investigate the pharmacologic and developmental regulation of the murine embryonic globin gene, epsilon y, in a homologous milieu in cell culture and in transgenic mice. Initial results have indicated that epsilon y gene expression can be up- regulated in a murine adult erythroid cell-line following differentiation with SCFAs; this is in sharp distinction to the lack of epsilon y gene expression in cells differentiated with DMSO. Preliminary analyses have implicated flanking sequences in the pharmacologic up-regulation and intragenic and/or downstream sequences in the DMSO-maintained silencing of epsilon y. Functional analyses of these identified sequences will be carried out in ES cells, and then transgenic mice to gain a full understanding of their physiologic role; ES cell transfectants and progeny of mice made transgenic for marked epsilon y globin gene constructs, with and without appropriate mutations or deletions, will be evaluated as to the developmental regulation of endogenous and transgenic ey globin gene expression; in addition, attempts will be made to pharmacologically re-activate transfected gene expression in adult transgenic progeny. It is anticipated that these studies will highlight potential future strategies for pharmacologic intervention and gene targeting therapy in human beta-globin gene disorders; in addition, it is expected that they will shed light on fundamental mechanisms of developmental gene regulation.